A new structure from cardiac cells cultured in vitro: Cardiomyocyte-annulation of neonatal rats

To explore the formation, morphological characteristics, cell composition, and differentiation potential of cardiomyocyte annulation (cardio-annulation) during in vitro culture of cardiac cells. Cardiac cells were isolated and cultured. A live-cell imaging system was used to observe cardio-annulation. Cardiac troponin-T (cTnT) and vimentin were labeled with double immunofluorescence staining, and coexpressions of cTnT and connexin43 (Cx43), cTnT and nanog, c-kit and nanog, and c-kit and stem cell antigen (sca-1) were detected. The location of various types of cells within the cardio-annulation structure was observed. Adipogenic- and osteogenic-inducing fluids were used separately for in situ induction to detect the multidirectional differentiation potential of cells during the annulation process. After 3 to 6 days, cardiac cells migrated and formed an open or closed annulus with a diameter of 800 to 3500 μm. The annulus wall comprised the medial, middle, and lateral regions. The cells in the medial region were small, abundant, and laminated, while those in the middle region were larger with fewer layers, and those in the lateral region were less abundant, and loosely arranged in a single layer. Cardiomyocytes were distributed mainly on the surface of the medial region; nanog⁺, c-kit⁺, and sca-1⁺ cells were located mainly at the bottom of the annulus wall and fibroblasts were located mainly between these layers. The annulus cavity contained a large number of small, round cells, and telocytes. Cx43 was expressed in all cell types, and nanog, c-kit, and sca-1 were coexpressed in the cardio-annulation cells, which possess adipogenic and osteogenic differentiation potential. Cardio-annulation was discovered during an in vitro culture of cardiac cells. The structure contains cardiomyocytes, fibroblasts, telocytes, and abundant stem cells. These results provide insight into the relationship among cardiac cells in vitro.     

Interactions of cardiac glycosides with cells and membranes. IV. Effects of ouabain and bumetanide on 86Rb+ influx in cultured cardiac myocytes from neonatal rats

Ouabain at nanomolar concentrations stimulates total Rb+ influx by 20 +/- 2% in monolayer cultures of myocytes which were either in physiologic ionic steady-state conditions ('control') or 'loaded with Na+' following exposure to K+-free medium. The ouabain-stimulated Rb+ influx was completely abolished by 0.1 mM bumetanide both in 'control' and in 'Na+-loaded' myocytes. Thus, addition of nanomolar concentrations of ouabain to myocytes markedly stimulate the bumetanide-sensitive Rb+ influx. This influx was increased up to 3- and 4-fold in 'control' and 'Na+-loaded' myocytes, respectively. Ouabain at nanomolar concentrations had no significant effect on the component of 86Rb+ influx which is inhibited by millimolar concentrations of ouabain (the so called 'ouabain-sensitive' or 'pump-mediated' Rb+ influx) in 'control' and 'Na+-loaded' cells. It is proposed that the increased rates of bumetanide-sensitive Rb+ influx are accompanied by an increased bumetanide-sensitive Na+ influx through the Na+/K+ cotransporter and thus to a transient increase in intracellular Na+ concentrations [Na+]i. The increase in [Na+]i, subsequently causes a transient elevation in [Ca2+]i via the Na+/Ca2+ exchanger and may be involved in the regulation of cardiac cells' contractility.     

Compensated cardiac hypertrophy is characterised by a decline in palmitate oxidation

Cardiac hypertrophy is an independent risk factor in the development of heart failure. However, the cellular mechanisms underlying the transition from compensated hypertrophy to heart failure are incompletely understood. The aim of this study was to investigate changes in myocardial substrate utilisation and function in pressure-overload hypertrophy (using ¹³C NMR spectroscopy) in parallel with alterations in the expression pattern of genes involved in cardiac fatty acid and glucose uptake and oxidation. Left ventricular hypertrophy was induced surgically in Sprague–Dawley rats by inter-renal aortic constriction. Nine weeks later, hearts were perfused in the isovolumic mode with a physiological mixture of substrates including 5 mM 1-¹³C glucose, 1 mM 3-¹³C lactate, 0.1 mM U-¹³C pyruvate and 0.3 mM U-¹³C palmitate and cardiac function monitored simultaneously. Real-time PCR was used to determine mRNA levels of PPARα and PPARα-regulated metabolic enzymes. Results showed that at the stage of compensated hypertrophy, fatty acid oxidation (FAO) and expression of genes involved in FAO were markedly reduced, whilst pyruvate oxidation was enhanced, highlighting the fact that metabolic remodelling is an early event in the development of cardiac hypertrophy.     

Abnormal fatty acid utilization by cultured cardiac cells from 7-day-old obese Zucker rats

Fatty acid utilization by muscle and nonmuscle heart cells in culture has been investigated in the 7-day-old Zucker rat to determine if this tissue could contribute to the lower energy expenditure reported in obese rats at the onset of obesity. The partitioning of oleate to oxidation and esterification products and the effect of genotype on this partitioning according to cell types were studied. Results showed that the fatty acid beta-oxidation and its esterification in neutral lipid was decreased by 30% in beating muscle cells from obese animals when compared with those from lean animals. In contrast, nonmuscle cells exhibited a decreased beta-oxidation alone. A similar fatty acid composition of the phospholipids was found in non-muscle cells of obese animals and their lean litter mates. In muscle cultures, palmitic and oleic acids are lower in cells of obese rats than in those of lean rats. The present study indicates that a defect in energy metabolism could be found in heart cells at the onset of obesity, suggesting that this defect is determined by intrinisic factor(s).     

Functional reentry in cultured monolayers of neonatal rat cardiac cells

Previous studies of reentrant arrhythmias in the heart have been performed in computer models and tissue experiments. We hypothesized that confluent monolayers of cardiac cells can provide a simple, controlled, and reproducible experimental model of reentry. Neonatal rat ventricular cells were cultured on 22-mm-diameter coverslips and stained with the voltage-sensitive dye RH-237. Recordings of transmembrane potentials were obtained from 61 sites with the use of a contact fluorescence imaging system. An electrical field stimulus, followed by a point stimulus, induced 39 episodes of sustained reentry and 21 episodes of nonsustained reentry. Sustained reentry consisted of single-loop (n = 18 monolayers) or figure-of-eight (n = 4) patterns. The cycle length, action potential duration at 80% repolarization, and conduction velocity were (in means +/- SE) 358 +/- 33 ms, 118 +/- 12 ms, and 12.9 +/- 1.0 cm/s for single loop and 311 +/- 78 ms, 137 +/- 18 ms, and 7.8 +/- 1.3 cm/s for figure-of-eight, respectively. Electrical termination by 6- to 13-V/cm field pulses or 15- to 20-V point stimuli was successful in 60% of the attempts. In summary, highly stable reentry can be induced, sustained for extensive periods of time, and electrically terminated in monolayers of cultured neonatal rat cardiac myocytes.     

Ionic determinants of spontaneous activity in clusters of cultured cardiac cells from newborn rats.

The spontaneous activity of cell clusters derived from ventricle cells of newborn rats was studied using a recording television microscope. The influence of varying concentrations of sodium, potassium, calcium, tetrodotoxin (TTX), and that of 2 mM MnCl2 was tested. The spontaneous activity of the cell clusters persisted in TTX but it was abolished by Mn. The beating rate increased when [Ca]0 and [Na]0 were changed from 0.3 mM to 3.0 mM and from 30 mM to 75 mM; it decreased with a change of [Na]0 from 75 mM to 142 mM. It is concluded that electrogenesis in their behavior to very young embryonic rat heart cells or cells of the rabbit sinoauricular node.     

Glucose oxidation by adherent and mechanically detached cultured cells.

This study examined the effect of mechanical detachment from the growth surface on energy metabolism of cultured cells. Oxidation of [1(-14)C]glucose measured by production of 14CO2 by adherent neuroblastoma (123 +/- 5 nmol/mg protein per minute), glioma (128 +/- 10 nmol/mg protein per minute), and fibroblast (137 +/- 5 nmol/mg protein per minute) cultures was similar. Removing cells from the culture flask by scraping reduced glucose oxidation by 62, 30, and 82% in neuroblastoma, glioma, and fibroblast cultures, respectively. Transferring cells from a culture flask to a test tube, to control for diffusional surface area, did not further reduce glucose oxidation. Detaching cells from the growth surface destroyed the extensive process formation and disrupted the normal spatial organization on the culture plate. These results indicate that it is essential to maintain these aspects of cellular architecture when evaluating metabolic properties of cultured cells.     

Metabolism of palmitate in cultured rat Sertoli cells

Isolated rat Sertoli cells were incubated in the presence of [1-14C]palmitate at a cell concentration of 1.54 +/- 0.31 mg protein/flask (n = 7). The oxidation of palmitate was concentration dependent and maximal oxidation was obtained at 0.35 mM-palmitate. At a saturating concentration of palmitate the oxidation was linear for at least 6 h. About 65% of the total amount of palmitate oxidized during 5 h at 0.52 mM-palmitate (109 +/- 44 nmol/flask, n = 5) was recovered as CO2 and the rest as acid-soluble compounds. Almost all radioactive acid-soluble compounds which were secreted by the Sertoli cells were shown to be 3-hydroxybutyrate and acetoacetate. The palmitate recovery in cellular lipids and triacylglycerols was 9.4 +/- 5.1 nmol/flask (n = 5) and 3.5 +/- 2.8 nmol/flask (n = 5) respectively. Addition of glucose had no significant effect on palmitate oxidation but caused a 9-fold increase in esterification of palmitate into triacylglycerols. We conclude that cultured rat Sertoli cells can oxidize palmitate to CO2 and ketone bodies and that fatty acids appear to be a major energy substrate for these cells.     

Contribution of Nav1.1 to cellular synchronization and automaticity in spontaneous beating cultured neonatal rat ventricular cells

Two factors of cell coupling influence cellular synchronization and automaticity: gap junction coupling and ion channels activity. However, the role of Na(+) channel isoforms underlying cell-to-cell interaction and cellular automaticity is not well understood. To address these questions, we studied mRNA expression of Na(+) channel isoforms and the effects of TTX on spontaneously beating cultured ventricle cells. Using RT-PCR technique we demonstrated the presence of Na(v)1.1 and Na(v)1.5 channels. The reduction of Na(v)1.1 channel activity disturbed cell-to-cell interaction and changed beating rates. Thus, Na(v)1.1 channel is involved in cellular synchronization and automaticity.     

Sarcoplasmic reticulum and intermediate filament organization in cultured neonatal cardiac muscle cells

Cardiac muscle cells from 3-day-old rat neonates were cultured for periods of 2 to 56 days. In order to facilitate ultrastructural studies on the organization of the sarcoplasmic reticulum, the cells were prepared for transmission electron microscopy according to a regimen including postfixation in reduced osmium ferrocyanide. The nonjunctional sarcoplasmic reticulum (NJSR) was organized as a loose, fenestrated sleeve around the exterior of bundles of myofilaments and was particularly prominent at the level of the Z line. The only recognizable junctional elements of the sarcoplasmic reticulum were in a peripheral location. Reduced osmium ferrocyanide was also useful in distinguishing intermediate (10 nm) filaments, since it understained Z substance, which often obscured these structures. Intermediate filaments were arranged both at the Z line and the intercalated disc, in parallel strands, approximately at right angles to the myofilaments.     

Urotensin II induces hypertrophic responses in cultured cardiomyocytes from neonatal rats.

Urotensin II (UII), a cyclic neuropeptide, functions not only in the central nervous system but also in non-neural systems including cardiovascular systems. In the present study we examined whether UII regulates hypertrophy in cardiomyocytes. The exposure of cultured cardiomyocytes from neonatal rats to UII dose-dependently activated extracellular signal-regulated kinases (ERKs), important molecules in the development of cardiac hypertrophy. ERK activation by UII at 100 nM peaked at 8 min after stimulation. UII markedly induced expression of specific genes encoding atrial natriuretic peptide and brain natriuretic peptide, and significantly increased amino acid incorporation into proteins. Incubation of cardiomyocytes with UII increased cell size and myofibril organisation. UII, then, might participate in cardiomyocyte hypertrophy.     

Ultrastructural morphometric analysis of cultured neonatal and adult rat ventricular cardiac muscle cells

Neonatal and adult rat ventricular cardiac muscle cells cultured on laminin differed from similar myocytes grown on plastic in the amount and distribution of their mitochondria and transverse tubules. Point-count morphometry was used at the electron microscopic level to quantify these differences. Adult myocytes grown on laminin contained more mitochondria per unit volume than adult myocytes grown on plastic. No significant differences were observed in the volume percent of myofibrils in either adult or neonatal ventricular myocytes when grown on laminin and compared to those grown on plastic. The transverse tubule system in neonatal and adult myocytes was reduced significantly when both groups were cultured on laminin. Furthermore, neonatal and adult myocytes cultured on laminin were flatter than those cultured on plastic. This may indicate a relationship between the surface/volume ratio and transverse tubule development in cultured myocytes. These studies establish that point-count morphometry can be used to quantify changes in the organelle volume densities of cultured cardiac muscle cells.     

Gene printer: laser-scanning targeted transfection of cultured cardiac neonatal rat cells

Heterogeneous gene expression in cardiac cells and tissues which requires targeted delivery of foreign DNA into selected cells or regions is needed for the development of novel therapies. Several techniques have been employed for targeted transfection, such as direct microinjection into cells or targeted electroporation. However, these techniques have limited bandwidth or spatial resolution of transfection. We aimed to develop a method for transfection of cardiac cells by means of laser-assisted optoporation using a standard confocal microscope. This technique allows for the transfection of selected cell types in the presence of other cell types as long as they are distinguishable with a microscope. This technique can work as a “gene printer” creating arbitrarily shaped areas of transfected cells.     

Glycolysis inhibition by palmitate in renal cells cultured in a two-chamber system

A major shortcoming of renalproximal tubular cells (RPTC) in culture is the gradual modification oftheir energy metabolism from the oxidative type to the glycolytic type.To test the possible reduction of glycolysis by naturally occurringlong-chain fatty acids, RPTC were cultured in a two-chamber system,with albumin-bound palmitate (0.4 mM) added to the basolateral chamberafter confluency. Twenty-four hours of contact with palmitate decreasedglycolysis by 38% provided that carnitine was present;lactate production was decreased by 38%, and the decrease inglycolysis resulted from a similar decrease of basolateral and apicalnet uptake of glucose. In contrast to the previously described effectof the nonphysiological oxidative substrate heptanoate, palmitatepromoted a long-term decrease in lactate production and sustainedexcellent cellular growth. After 4 days of contact, decreasedglycolysis was maintained even in the absence of carnitine and resultedfrom a decrease of basolateral uptake only, suggestive of long-term regulation different from the earlier effects. Thus, although culturedRPTC lost their oxidative phenotype, they exhibited a type ofregulation (Randle effect) that is found in the oxidative-type but notin the glycolytic-type tissues, therefore unmasking a regulativecapacity barely detectable in fresh RPTC. LowPO₂ (50 mmHg in the apical chamber) could be amajor cause of elevated glycolysis and could hinder the effects ofpalmitate.